EXPERIMENTAL AND THERAPEUTIC MEDICINE 19: 467-472, 2020

Effects of combined with metformin on serum cystatin C, homocysteine and maternal and neonatal outcomes in pregnant women with gestational diabetes mellitus

JIZENG ZHENG1, JUAN XU2, YIN ZHANG1 and NAN ZHOU3

1Department of Obstetrics and Gynecology, Liaocheng Third People's Hospital, Liaocheng, Shandong 252000; 2Department of Obstetrics, Tengzhou Central People's Hospital, Tengzhou, Shandong 277500; 3Department of Health Care, Jinan Central Hospital, Jinan, Shandong 250013, P.R. China

Received July 5, 2019; Accepted September 2, 2019

DOI: 10.3892/etm.2019.8224

Abstract. Effects of insulin combined with metformin on Introduction serum cystatin C (Cys C), homocysteine (Hcy) and maternal and neonatal outcomes in pregnant women with gestational Gestational diabetes mellitus (GDM) (1,2) is a disease that diabetes mellitus (GDM) were investigated. In total, 80 cases occurs during pregnancy, and its incidence rate has gradually of pregnant women diagnosed with GDM in the Department increased. Lack of pregnancy knowledge, along with unrea- of Obstetrics and Gynecology of Liaocheng Third People's sonable diet, glycemic control and pregnancy weight gain lead Hospital from July 2015 to July 2017 were selected and divided to GDM in pregnant women (3). These are important factors into a study group (42 cases) and a control group (38 cases). in becoming a high‑risk pregnant woman. If not controlled The study group was treated with insulin combined with in time, the disease may have a severe impact on mothers metformin, and the control group was treated with insulin. and infants. Fasting blood glucose (FBG) and postprandial blood glucose Most pregnant women with GDM cannot control their after 2 h (2hPG) of the two groups were compared before and blood glucose effectively through diet control and exercise after treatment. Levels of serum Cys C, Hcy, urinary protein therapy (4). Consequently, they are often dependent on drug (UmAlb), postpartum maternal outcomes and adverse reac- therapy to control their blood glucose level. Insulin is often tions during pregnancy were compared in the two groups selected for the treatment of diabetes as it can reduce blood before and after treatment. After treatment, the level of FBG glucose levels (5). However, it has no obvious effect on the and 2hPG in the control group was higher than that in the treatment of insulin resistance in patients with GDM; thus, treatment group (P<0.05). After treatment, the level of serum there are still some deficiencies in its efficacy. Metformin is an Cys C and Hcy in both groups were lower than that before the insulin sensitizer that adjusts glucose metabolism by adjusting treatment, and the level in the study group was lower than that insulin sensitivity (6). This constitutes an important reason for in the control group (P<0.05). The total incidence of neonatal improving insulin sensitivity in vivo. adverse outcomes and the number of adverse pregnancies in Increasing insulin resistance in GDM patients is the GDM patients in the study group were significantly lower than result of increased secretion of , and those in the control group (P<0.05). There were no significant other substances in the middle and late stages of gestation, differences in adverse reactions during pregnancy between the resulting in decreased insulin sensitivity, unstable blood two groups (P>0.05). In conclusion, insulin combined with glucose level, and abnormal glucose metabolism and various metformin is more effective than insulin alone in reducing complications (7). Diabetic nephropathy is easy to occur serum Cys C and Hcy levels, with significant effect on the during the treatment of GDM (8). Due to the inconspicuous improvement of maternal and neonatal outcomes. early symptoms, diabetic nephropathy is not easy to detect, and develops to the point of severe damage when identified at late stage. While cystatin C (Cys C) is a sensitive indicator for early diagnosis of damage to glomerular filtration function, it can reflect the damage degree of patients with GDM compli- Correspondence to: Dr Nan Zhou, Department of Health Care, Jinan Central Hospital, 105 Jiefang Road, Jinan, Shandong 250013, cated with renal disease (9). In addition, when the body's P. R. Ch i na blood glucose level is at a high concentration, patients may E‑mail: [email protected] lose excessive amounts of fluid (10,11). The level of homo- cysteine (Hcy) also increases, accelerating the pathological Key words: insulin, metformin, serum cystatin C, homocysteine, changes of small blood vessels and aggravating the course maternal and neonatal outcomes of GDM (10,11). Therefore, the monitoring of the two serum factors is conducive to the prevention and timely detection of some related complications. 468 ZHENG et al: INSULIN COMBINED WITH METFORMIN ON Cys C AND Hcy IN GDM

Previous findings have shown that insulin combined with Maternal and neonatal outcomes between the two groups metformin have an effect of declining the levels of blood were compared, including maternal delivery methods: sugar (12). However, few studies on the joint treatment of the Cesarean section, preterm delivery and labor induction. two concerning the detection of serum Cys C and Hcy levels The number of newborns with hypoglycemia, macrosomia, in patients with GDM and their effects on mothers and infants neonatal jaundice, and fetal malformation was recorded. are currently available. In the present study, we explored the UmAlb levels of pregnant women in the two groups were effect of the combined therapy of insulin and metformin on compared, special protein immunoturbidimetry was used serum Cys C and Hcy levels, and the maternal and neonatal in the detection. The detection instrument was Beckman outcomes in GDM patients. DXC600 automatic biochemical detector (Beckmen Coulter). The incidence of adverse reactions was compared between Patients and methods the two groups, including nausea, vomiting, hypertension and diarrhea. General data. A total of 80 cases of GDM patients admitted to the department of obstetrics and gynecology of Liaocheng Statistical analysis. In this study, SPSS 20.0 [Boyi zhixun Third People's Hospital (Liaocheng, China) from July 2015 (Beijing) Information Technology Co., Ltd.[ was used for statis- to July 2017 were selected, 42 cases of patients treated with tical analysis of experimental data. Chi‑square test was used insulin combined with metformin were included in the study for enumeration data. Mean ± standard deviation was used for group, and 38 cases of patients treated with insulin alone measurement data. The t‑test was used for comparison between were included in the control group. There were no significant the two groups, and paired t‑test was used for comparison before differences in age, BMI and gestational weeks (P>0.05). and after treatment of the two groups. GraphPad Prism 6 soft- Patients who met one of the diagnostic criteria for GDM ware was used to draw the experimental illustrations. P<0.05 were included in the study: fasting blood glucose (FPG) was considered a statistically significant difference. ≥5.1 mmol/l, blood glucose after 1 h (1hPG) ≥10.0 mmol/l, and postprandial blood glucose after 2 h (2hPG) ≥8.5 mmol/l (13). Results Exclusion criteria were: patients who had gestational diabetes during previous pregnancy; patients with liver and kidney Comparison of general data of the two groups. There were no dysfunction; patients with communication and cognitive significant differences in general data such as age, BMI and dysfunction; patients who did not cooperate with the study gestational weeks (P>0.05) (Table I). requirements. All the patients and their families agreed to participate in Comparison of FBG and 2hPG levels before and after treat- the experiment and signed an informed consent. The present ment in the two groups. The FBG content in the study group study was approved by the Ethics Committee of the Liaocheng and the control group before treatment was 6.75±0.68 and Third People's Hospital. 6.72±0.67 mmol·l‑1, and the 2hPG content was 10.34±2.45 and 10.27±2.23 mmol·l‑1, respectively. After treatment, the content Therapeutic schedule. Regular exercise and diet management of FBG was 5.13±0.46 and 5.67±0.52 mmol·l‑1, respectively. were adopted in both groups. Patients in the control group The 2hPG content was 5.24±2.56 and 6.12±2.67 mmol·l‑1, received insulin treatment [import drug registration certificate respectively. There were no significant differences between no. X19990279[96], medicine authorized number: J‑70[2]; the study and control groups before treatment (P>0.05), and Novo Nordisk (China) Pharmaceuticals Co., Ltd.], at the dose the levels of FBG and 2hPG in the study group after treatment of 0.2-0.25 U/(kg·day). Patients in the study group received were lower than those in the control group (P<0.05) (Fig. 1). treatment of insulin combined with metformin. The insulin dose was 0.2-0.25 U/(kg·day), and the metformin was given Content of serum Cys C and Hcy of patients in the two groups orally at 0.5 g/time twice a day. The dosage could be increased before and after treatment. Serum Cys C contents in the or decreased according to the patient's blood glucose level, study group and control group before treatment were 1.480.56 and the highest dose could not exceed 3 g/day. The treatment and 1.460.45 mg/l, respectively. The contents of serum Hcy continued until the birth of the newborn. were 14.25 and 14.285.34 µmol/l, respectively. There were no significant differences between the two groups (P>0.05). After Observational indexes. Blood glucose levels of GDM patients treatment, serum Cys C levels in the study group and control in both groups were observed before and after treatment, and group were 0.780.34 and 1.210.25 mg/l, respectively. The FBG and 2hPG levels were measured and recorded in all serum Hcy levels were 7.212.67 and 103.48 µmol/l, respec- patients before and after treatment. tively. After treatment, the levels of both groups were lower Venous blood (5 ml) of the pregnant women was extracted than those before treatment, but the serum levels of Cys C and before and after treatment and centrifuged at 2,600 x g for Hcy in the study group were significantly lower than those 10 min at 4˚C. Then, the serum was placed in the refrig- in the control group, with statistically significant differences erator (AU5800 automatic biochemical analyzer; Beckmen (P<0.05) (Fig. 2). Coulter). ELISA was used to detect the level of Hcy in serum (EY‑elisa2279; Shanghai Yiyan Biological Technology Co., Comparison of urinary protein levels before and after treat- Ltd.). Immunoturbidimetry was used to detect the serum ment in the two groups. Urine protein levels in the study Cys C level (59400368211; Hangzhou Deangel Biological group and control group before treatment were 17.271.68 and Engineering Co., Ltd.). 17.451.64 mg/mmol, respectively, and there were no significant EXPERIMENTAL AND THERAPEUTIC MEDICINE 19: 467-472, 2020 469

Table I. Comparison of general data of the two groups.

Factors Research group (n=42) Control group (n=38) t/χ2 value P-value

Age (years) 0.179 0.673 ≤26 28 (66.67) 27 (71.05) >26 14 (33.33) 11 (28.94) Family history 0.038 0.846 With 5 (11.90) 4 (10.52) Without 37 (88.10) 34 (89.47) Embryo number 0.011 0.918 Single birth 40 (95.24) 36 (94.74) Twins 2 (4.76) 3 (7.89) History of abortion 0.506 0.613 With 8 (19.05) 9 (23.68) Without 34 (80.95) 29 (76.32) BMI before pregnancy (kg/m2) 21.45±0.38 21.83±0.26 0.111 0.739 Body growth during gestation (kg) 16.24±0.27 16.34±0.34 0.128 0.617 Gravidity 1.67±0.38 1.58±0.25 0.220 1.237 Parity 1.35±0.26 1.46±0.37 0.125 1.550 Gestational weeks 27.47±2.27 27.42±1.82 0.108 0.914

Figure 1. Comparison of FBG and 2hPG levels before and after treatment in the two groups. (A) FBG levels in both the study and control groups were significantly reduced after treatment, but the reduction level in the study group was higher than that in the control group. (B) Τhe 2hPG level in both the study and control groups was significantly reduced after treatment, but the reduction level in the study group was higher than that in the control group. *P<0.05. FBG, fasting blood glucose; 2hPG, postprandial blood glucose after 2 h. differences between the two groups (P>0.05). After treatment, vomiting, dizziness, diarrhea and hypertension in the control the urine protein levels of the study group and the control group were 1, 1, 0 and 1, respectively. There were no significant group were 9.781.21 and 12.861.45 mg/mmol, respectively. differences in the total number of adverse reactions between After treatment, the urine protein levels of both groups were the two groups (P>0.05) (Table II). All adverse reactions were lower than those before treatment, and the level in the study relieved after symptomatic treatment. group was significantly lower than that in the control group, with statistically significant differences (P<0.05) (Fig. 3). Comparison of neonatal outcomes between the two groups. When pregnancy ended, the number of neonates with hypogly- Comparison of adverse reactions during pregnancy between the cemia, neonatal jaundice, macrosomia and fetal abnormalities two groups. The number of patients with nausea and vomiting, in the study group were 1, 2, 2 and 1, respectively. The number dizziness, diarrhea and hypertension in the study group were 2, of the neonates with those symptoms in the control group were 1, 2 and 1, respectively. The number of patients with nausea and 3, 5, 3 and 2, respectively. The incidence of neonatal adverse 470 ZHENG et al: INSULIN COMBINED WITH METFORMIN ON Cys C AND Hcy IN GDM

Figure 2. Content of serum Cys C and Hcy of patients in the two groups before and after treatment. (A) Serum Cys C levels in both the study and control groups were significantly reduced after treatment, but the reduction level in the study group was higher than that in the control group. (B) Serum Hcy levels in both the study and control groups were significantly reduced after treatment, but the reduction level in the study group was greater than that in the control group. *P<0.05. Cys C, cystatin C; Hcy, homocysteine.

on human health. However, the pathogenesis of GDM is still unknown. GDM is known to cause great harm to pregnant women and infants (15). Thus, the primary principle of GDM treatment is to control the blood glucose level of GDM patients to a normal level, so as to reduce the occurrence of related complications of pregnant women and infants. Metformin is the primary choice for the treatment of type 2 diabetes for its significant efficacy in controlling blood glucose level, with low incidence of hypoglycemia and protective effect on cardiovascular system (16). It is therefore widely used in clinical practice. In addition, metformin is often used alone or in combination with other drugs in clinical practice (17). Therefore, in the present study, we used insulin combined with metformin to treat GDM patients. Comparisons of the levels of FBG and 2hPG before and after treatment in the study group and the control group showed reduced level in the study group Figure 3. Comparison of urinary protein levels before and after treatment treated with insulin combined with metformin was greater between the two groups. The urine protein levels of both the study group and than that in the control group treated with insulin alone. There the control group were significantly reduced after treatment, but the decrease were no significant differences in adverse reactions during * level of the study group was greater than that of the control group. P<0.05. pregnancy in the two groups, suggesting that insulin combined with metformin was more effective in lowering blood sugar levels and did not increase the incidence of adverse reactions outcomes in the study group were significantly lower than during pregnancy. It confirmed the safety of the two drugs. those in the control group (P<0.05) (Table III). Studies have also confirmed that insulin combined with insulin sensitizers has a relatively significant effect on the control Comparison of adverse pregnancy outcomes in the two of blood glucose level in the body, and can also reduce the groups. The number of adverse pregnancies in the study group incidence of maternal and neonatal complications (18). Other was 10, and the number of adverse pregnancies in the control studies have shown that insulin and metformin are safe and group was 17. The number of adverse pregnancies in the study effective during pregnancy (19). Those findings are consistent group was significantly lower than that in the control group with our results. (P<0.05) (Table IV). Cys C is mainly filtered and cleared in the kidneys (20,21). If the glomerulus of the body is damaged, its level will Discussion increase rapidly and be positively correlated with the degree of injury (20,21). Hcy is one of the risk factors of vasculopathy (22). With the development of the society, people's quality of life When vascular endothelial injury occurs, the risk of diabetic has improved, and the incidence of GDM (14) has increased nephropathy increases. Studies have shown that serum levels of with the constant change of dietary forms. A high proportion Cys C and Hcy are somewhat correlated with disease progres- of the incidence among young people has lead to an impact sion and maternal and neonatal complications (23). Therefore, EXPERIMENTAL AND THERAPEUTIC MEDICINE 19: 467-472, 2020 471

Table II. Comparison of adverse reactions during pregnancy in the two groups.

Adverse reactions Research group (n=42) Control group (n=38) χ2 value P-value

Nausea and vomiting 2 (4.76) 1 (2.63) 0.251 0.617 Dizziness 1 (2.38) 1 (2.63) 0.005 0.942 Diarrhea 2 (4.76) 0 (0) 1.856 0.173 Hypertension 1 (2.38) 2 (5.36) 0.459 0.498 Total number 6 (14.27) 4 (10.53) 0.612 0.258

Table III. Comparison of neonatal outcomes between the two groups.

Groups Research group (n=42) Control group (n=38) χ2 value P-value

Hypoglycemia 1 (2.08) 3 (6.25) 1.277 0.259 Neonatal jaundice 2 (4.16) 3 (7.89) 1.761 0.185 Macrosomia 3 (7.14) 5 (13.15) 0.334 0.563 Fetal abnormalities 1 (2.08) 2 (5.26) 0.459 0.498 Total number 7 (16.67) 13 (34.21) 4.373 0.037

Table IV. Comparison of adverse pregnancy outcomes in the two groups.

Groups Research group (n=42) Control group (n=38) χ2 value P-value

Cesarean section 2 (4.76) 4 (10.53) 0.956 0.328 Premature birth 4 (9.52) 5 (13.16) 0.264 0.608 Polyhydramnios 2 (4.76) 4 (10.53) 0.956 0.328 Premature rupture of membrane 1 (2.38) 2 (5.23) 0.460 0.498 Postpartum hemorrhage 1 (2.38) 2 (5.23) 0.460 0.498 Total number 10 (23.81) 17 (44.73) 3.908 0.048

we compared the levels of the patients in the study and control outcomes (25). In addition, maternal and neonatal outcomes groups before and after treatment. The results showed that of GDM depend on blood glucose control, and good blood there were no significant differences in serum Cys C and Hcy glucose control can reduce the incidence of maternal and levels between the two groups before treatment, and the levels neonatal complications (26). Therefore, we compared the of patients in the study group after treatment were lower than number of neonatal hypoglycemia, neonatal jaundice, macro- those in the control group. At the same time, in order to study somia and fetal malformation in the two groups, and found its correlation with renal injury, we also compared the urine that the total number of adverse outcomes in the study group protein levels of the two groups before and after treatment. It was lower than that in the control group, and the total number was found that there were no significant differences between of adverse pregnancies in the GDM patients in the study the urine protein levels of the two groups before treatment. group was significantly lower than that in the control group. However, the urine protein levels of the treatment group were This finding indicated that insulin combined with metformin significantly lower than those of the control group. Therefore, had a significant effect on reducing blood glucose level in it was speculated that the increase of serum Cys C and Hcy GDM patients, which could reduce adverse pregnancy and levels would lead to an increase of urinary protein levels, fetal outcomes in pregnant women, and it was conducive to which may affect the kidneys of GMD pregnant women to improving maternal and neonatal quality of life. Therefore, some extent. The above results suggest that insulin combined effective control of blood glucose level is necessary in GDM. with metformin can effectively reduce the serum Cys C and In conclusion, the use of insulin combined with metformin Hcy levels of patients, thus improving the patient's condition. can effectively reduce the levels of FBG, 2hPG and serum Previous findings have indicated that the therapeutic effect Cys C and Hcy in GDM patients, and can effectively improve and complications of patients with GDM could be predicted maternal and neonatal outcomes without increasing adverse by monitoring factors in serum of patients with GDM (24). reactions during pregnancy. However, the current study did not GDM not only affects the normal development of infants, make a comparison, for example, with other types of insulin, but also greatly increases the incidence of adverse pregnancy making further studies necessary. 472 ZHENG et al: INSULIN COMBINED WITH METFORMIN ON Cys C AND Hcy IN GDM

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